Assays and other reactions involving droplets

ABSTRACT

The present invention generally relates to droplets and/or emulsions, such as multiple emulsions. In some cases, the droplets and/or emulsions may be used in assays, and in certain embodiments, the droplet or emulsion may be hardened to form a gel. In some aspects, a heterogeneous assay can be performed using a gel. For example, a droplet may be hardened to form a gel, where the droplet contains a cell, DNA, or other suitable species. The gel may be exposed to a reactant, and the reactant may interact with the gel and/or with the cell, DNA, etc., in some fashion. For example, the reactant may diffuse through the gel, or the hardened particle may liquefy to form a liquid state, allowing the reactant to interact with the cell. As a specific example, DNA contained within a gel particle may be subjected to PCR (polymerase chain reaction) amplification, e.g., by using PCR primers able to bind to the gel as it forms. As the DNA is amplified using PCR, some of the DNA will be bound to the gel via the PCR primer. After the PCR reaction, unbound DNA may be removed from the gel, e.g., via diffusion or washing. Thus, a gel particle having bound DNA may be formed in one embodiment of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/529,926, with a §371(c) date of Feb. 10, 2010, which is a nationalstage filing under 35 U.S.C. §371 of international applicationPCT/US2008/003185, filed Mar. 7, 2008, which claims benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No.60/905,567, filed Mar. 7, 2007, the contents of each of which areincorporated herein by reference in their entities.

FIELD OF INVENTION

The present invention generally relates to droplets and/or emulsions. Insome cases, the droplets and/or emulsions may be used in assays.

BACKGROUND

An emulsion is a fluidic state which exists when a first fluid isdispersed in a second fluid that is typically immiscible orsubstantially immiscible with the first fluid. Examples of commonemulsions are oil in water and water in oil emulsions. Multipleemulsions are emulsions that are formed with more than two fluids, ortwo or more fluids arranged in a more complex manner than a typicaltwo-fluid emulsion. For example, a multiple emulsion may beoil-in-water-in-oil, or water-in-oil-in-water. Multiple emulsions are ofparticular interest because of current and potential applications infields such as pharmaceutical delivery, paints and coatings, food andbeverage, and health and beauty aids.

Typically, multiple emulsions consisting of a droplet inside anotherdroplet are made using a two-stage emulsification technique, such as byapplying shear forces through mixing to reduce the size of dropletsformed during the emulsification process. Other methods such as membraneemulsification techniques using, for example, a porous glass membrane,have also been used to produce water-in-oil-in-water emulsions.Microfluidic techniques have also been used to produce droplets insideof droplets using a procedure including two or more steps. For example,see International Patent Application No. PCT/US2004/010903, filed Apr.9, 2004, entitled “Formation and Control of Fluidic Species,” by Link,et al., published as WO 2004/091763 on Oct. 28, 2004; or InternationalPatent Application No. PCT/US03/20542, filed Jun. 30, 2003, entitled“Method and Apparatus for Fluid Dispersion,” by Stone, et al., publishedas WO 2004/002627 on Jan. 8, 2004, each of which is incorporated hereinby reference. See also Anna, et al., “Formation of Dispersions using‘Flow Focusing’ in Microchannels,” Appl. Phys. Lett., 82:364 (2003) andOkushima, et al., “Controlled Production of Monodispersed Emulsions byTwo-Step Droplet Breakup in Microfluidic Devices,” Langmuir 20:9905-9908(2004). In some of these examples, a T-shaped junction in a microfluidicdevice is used to first form an aqueous droplet in an oil phase, whichis then carried downstream to another T-junction where the aqueousdroplet contained in the oil phase is introduced into another aqueousphase. In another technique, co-axial jets can be used to produce coateddroplets, but these coated droplets must be re-emulsified into thecontinuous phase in order to form a multiple emulsion. See Loscertaleset al., “Micro/Nano Encapsulation via Electrified Coaxial Liquid Jets,”Science 295:1695 (2002).

Multiple emulsions and the products that can be made from them can beused to produce a variety of products useful in the food, coatings,cosmetic, or pharmaceutical industries, for example. Methods forproducing multiple emulsions providing consistent droplet sizes,consistent droplet counts, consistent coating thicknesses, and/orimproved control would make commercial implementation of these productsmore viable.

SUMMARY OF THE INVENTION

The present invention generally relates to droplets and/or emulsions. Insome cases, the droplets and/or emulsions may be used in assays. Thesubject matter of the present invention involves, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of one or more systems and/orarticles.

In one aspect, the invention is a method. In one set of embodiments, themethod includes acts of providing a fluidic droplet containing aspecies, hardening the fluidic droplet containing the species, andexposing the species within the hardened fluidic droplet to a reactant.In another set of embodiments, the method includes acts of providing agel droplet comprising a first nucleic acid and a second nucleic aciddifferent from the first nucleic acid, the first nucleic acid beingbound to the gel, and growing the first nucleic acid within the gel,using the second nucleic acid as a template. In still another set ofembodiments, the method includes acts of providing a fluidic dropletcontaining a species, hardening the fluidic droplet containing thespecies, and liquefying the hardened fluidic droplet. In another set ofembodiments, the method includes acts of providing a first fluidicdroplet and a second fluidic droplet, and causing the first fluidicdroplet and the second fluidic droplet to fuse, wherein the fuseddroplet hardens.

The method, in yet another set of embodiments, includes hardening afluidic droplet containing cells, and causing the cells within thehardened fluidic droplet to multiply.

In another set of embodiments, the method includes an act of causing aPCR reaction to occur within a gel droplet. In still another set ofembodiments, the method includes an act of forming a gel dropletcontaining a PCR primer bound to the gel.

In one set of embodiments, the method includes acts of providing afluidic droplet in a carrying fluid, the fluidic droplet substantiallyimmiscible in water and the carrying fluid substantially immiscible inwater, hardening the fluidic droplet, removing the carrying fluid, andplacing the hardened fluidic droplet in a third fluid.

In another aspect, the invention is an article. In one embodiment, thearticle includes a gel droplet containing a clonal population of cells.

In another embodiment, the article includes a gel droplet containing aPCR primer bound to the gel.

In another aspect, the present invention is directed to a method ofmaking one or more of the embodiments described herein. In anotheraspect, the present invention is directed to a method of using one ormore of the embodiments described herein.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-1D illustrates a microfluidic device of one embodiment of theinvention;

FIGS. 2A-2C illustrate the formation of droplets in accordance withanother embodiment of the invention;

FIGS. 3A-3C illustrate temperature dependence of various microgels, inyet another embodiment of the invention;

FIGS. 4A-4B illustrate the probing of DNA in various hydrogel particles,in another embodiment of the invention; and

FIG. 5 is a schematic illustration of a microfluidic device useful inmaking multiple emulsions, according to one embodiment of the invention.

DETAILED DESCRIPTION

The present invention generally relates to droplets and/or emulsions,such as multiple emulsions. In some cases, the droplets and/or emulsionsmay be used in assays, and in certain embodiments, the droplet oremulsion may be hardened to form a gel. In some aspects, a heterogeneousassay can be performed using the gel. For example, a droplet may behardened to form a gel, where the droplet contains a cell, DNA, or othersuitable species. The gel may be exposed to a reactant, and the reactantmay interact with the gel and/or with the cell, DNA, etc., in somefashion. For example, the reactant may diffuse through the gel, or thehardened particle (or a portion thereof) may liquefy to form a liquidstate, allowing the reactant to interact with the cell. As a specificexample, DNA contained within a gel particle may be subjected to PCR(polymerase chain reaction) amplification, e.g., by using PCR primersable to bind to the gel as it forms. As the DNA is amplified using PCR,some of the DNA will be bound to the gel via the PCR primer. After thePCR reaction, unbound DNA may be removed from the gel, e.g., viadiffusion or washing. Thus, a gel particle having bound DNA may beformed in one embodiment of the invention.

Thus, in one aspect, the invention involves reactions involving liquiddroplets, for example, droplets contained within emulsions such asmultiple emulsions. In certain embodiments, systems and methods areproviding for causing two or more droplets to fuse or coalesce, e.g., incases where the droplets ordinarily are unable to fuse or coalesce, forexample due to composition, surface tension, size, etc., e.g., to causea reaction to occur. For example, in a microfluidic system, the surfacetension of the fluidic droplets, relative to their size, may preventfusion of the fluidic droplets. The fluidic droplets may eachindependently contain gas or liquid.

Fields in which droplets and emulsions may prove useful include, forexample, food, beverage, health and beauty aids, paints and coatings,and drugs and drug delivery. For instance, a precise quantity of a drug,pharmaceutical, or other agent can be encapsulated by a shell designedto release its contents under particular conditions, as described indetail below. In some instances, cells can be contained within adroplet, and the cells can be stored and/or delivered. Other speciesthat can be stored and/or delivered include, for example, biochemicalspecies such as nucleic acids such as siRNA, RNAi and DNA, proteins,peptides, or enzymes. Additional species that can be incorporated withina droplet or emulsion of the invention include, but are not limited to,nanoparticles, quantum dots, fragrances, proteins, indicators, dyes,fluorescent species, chemicals, or the like. A droplet or emulsion canalso serve as a reaction vessel in certain cases, such as forcontrolling chemical reactions, or for in vitro transcription andtranslation, e.g., for directed evolution technology.

Using the methods and devices described herein, in some embodiments, aconsistent size and/or number of droplets can be produced, and/or aconsistent ratio of size and/or number of outer droplets to innerdroplets, inner droplets to other inner droplets, or other such ratios,can be produced. For example, in some cases, a single droplet within anouter droplet of predictable size can be used to provide a specificquantity of a drug. In addition, combinations of compounds or drugs maybe stored, transported, or delivered in a emulsion or droplet. Forinstance, hydrophobic and hydrophilic species can be delivered in asingle droplet, as the droplet can include both hydrophilic andhydrophobic portions. The amount and concentration of each of theseportions can be consistently controlled according to certain embodimentsof the invention, which can provide for a predictable and consistentratio of two or more species in the multiple droplet.

Thus, in one aspect, the present invention is directed to systems,assays, methods, etc. involving gels produced as described herein, e.g.,gel particles, gel capsules, and the like. In some cases, aheterogeneous assay involving the gel can be performed. A gel may alloweasier use of a heterogeneous assay than by using a liquid emulsion dropdirectly. That is, once a gel is formed, its composition can be changedby adding new reagents or washing out old ones.

One non-limiting example of a heterogeneous assay in a microgel particleis similar to a “polony,” as described in detail in U.S. Pat. Nos.6,432,360, 6,485,944 and 6,511,803, and in PCT/US05/06425, eachincorporated by reference, as well as U.S. patent application Ser. No.11/505,073, also incorporated herein by reference. Briefly, in oneembodiment, one or more DNA molecules (or other species, as describedherein), either naked, or contained within an intact cell, areencapsulated within a microgel at the time of formation, e.g., byforming a droplet containing the cell, DNA molecule, other species,etc., then hardening the droplet, e.g., to form a gel. In some cases,molecules of one or more PCR primers having 5′ acrydite moieties may beincluded in the droplet, prior to hardening. Upon polymerization of thegel, the primer may be covalently coupled to the gel matrix, via theacrydite. The emulsion droplets may thus be collected and caused topolymerize or gel. The surrounding fluid (e.g., an oil phase) can thenbe removed, e.g., by washing away with a suitable solvent, leaving gelparticles containing the cell, DNA molecule, other species, etc., andthe PCR primers. The gels can, in some cases, be resuspended in anaqueous solution containing reagents for PCR (e.g., bufferingcomponents, salts, dNTPs, DNA polymerase, and/or one or more PCR primerswith no acrydite modification). The suspension of gel particles can thenbe thermally cycled in a test tube, e.g., using standard PCR cyclingtechniques known to those of ordinary skill in the art, which may allowPCR amplification to occur, e.g., between pairs of primers. The strandof DNA synthesized from the acrydite-modified primers may thus becovalently coupled to the gel in some cases.

The gels can be washed to remove unreacted or unbound PCR components,including DNA strands synthesized from non-acrydite primers. Theremaining covalently attached DNA strand can be probed using techniquesknown to those of ordinary skill in the art, for example, by bindingsequence-specific fluorescent oligonucleotides and washing away unboundprobes. As another example, a single-base extension reaction can be usedto probe the DNA sequence at a particular site. The fluorescence of thegel from the bound oligonucleotides can be measured by measured usingtechniques known to those of ordinary skill in the art, such asfluorescence microscopy or by FACS. Either the presence or absence of asequence can be determined, and/or the sequence state at one or morepositions can be determined, for example, genotyping by SNPs. Forinstance, by using FACS, sub-populations of the gels can be sorted andanalyzed separately.

As discussed herein, in another set of embodiments, cells of any type(prokaryotic or eukaryotic) can be encapsulated in the gel, and the DNAor RNA within the cell can be used as a template for enzymaticamplification, in one embodiment of the invention. This could be done,for example, by reverse transcription PCR (rtPCR) for the study of RNAwithin the cell, or standard PCR for DNA. As another example, othertypes of enzymatic amplification, such as whole-genome amplification byphi29 polymerase, can also be performed.

As yet another example, a collection of gels with covalently attachedDNA may be used as a library, e.g., that could be probed and washed manytimes in succession, which would be especially useful when whole genomesare amplified in the gel, since many regions of the DNA could be probedover time. Furthermore, in some cases, by doing PCR from the librarygels using unmodified primers, chosen sections of DNA can be amplifiedaway from the gel and analyzed further. For example, a gene can beamplified from a sub-population of library gels and then the supernatantfrom the PCR could be sequenced by standard methods.

In another embodiment of the invention, particles, such as polymerbeads, may be encapsulated or incorporated into droplets which are thenhardened into gels (e.g., forming gel particles, gel capsules, etc.). Inone embodiment, the beads may be magnetic, which could allow for themagnetic manipulation of the gels. The beads could also befunctionalized so that they could have other molecules attached, such asproteins, nucleic acids or small molecules. One embodiment of thepresent invention is directed to a set of beads encoding a library of,for example, nucleic acids, proteins, small molecules, or other speciesas described herein, that would stay embedded in a gel particleindefinitely.

As mentioned, in certain aspects, the invention generally relates toemulsions and/or droplets. The emulsion may include droplets, such asthose described above, and/or colloid particles. As used herein, an“emulsion” is given its ordinary meaning as used in the art, i.e., aliquid dispersion. In some cases, the emulsion may be a “microemulsion”or a “nanoemulsion,” i.e., an emulsion having a dispersant on the orderof microns or nanometers, respectively. The dispersion or emulsion, insome cases, may include droplets having a homogenous distribution ofdiameters, i.e., the droplets may have a distribution of diameters suchthat no more than about 10%, about 5%, about 3%, about 1%, about 0.03%,or about 0.01% of the droplets have an average diameter greater thanabout 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% ofthe average diameter of the droplets. As one example, such an emulsionmay be created by allowing fluidic droplets of the appropriate size orsizes (e.g., created as described herein) to enter into a solution thatis immiscible with the fluidic droplets.

Techniques for forming droplets have been disclosed in, for example,U.S. patent application Ser. No. 11/360,845, filed Feb. 23, 2006,entitled “Electronic Control of Fluidic Species,” by Link, et al.,published as U.S. Patent Application Publication No. 2007/000342 on Jan.4, 2007, incorporated herein by reference. For example, electric fieldsmay be used to create droplets of fluid surrounded by a liquid. Thefluid and the liquid may be essentially immiscible in many cases, i.e.,immiscible on a time scale of interest (e.g., the time it takes afluidic droplet to be transported through a particular system ordevice). In certain cases, the droplets may each be substantially thesame shape or size, as further described below. The fluid may alsocontain other species, for example, certain molecular species (e.g., asfurther discussed below), cells, particles, etc.

The electric field, in some embodiments, is generated from an electricfield generator, i.e., a device or system able to create an electricfield that can be applied to the fluid. The electric field generator mayproduce an AC field (i.e., one that varies periodically with respect totime, for example, sinusoidally, sawtooth, square, etc.), a DC field(i.e., one that is constant with respect to time), a pulsed field, etc.The electric field generator may be constructed and arranged to createan electric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments. As used herein, “integral” meansthat portions of the components integral to each other are joined insuch a way that the components cannot be manually separated from eachother without cutting or breaking at least one of the components.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof. In some cases, transparent or substantiallytransparent electrodes can be used.

In some embodiments of the invention, systems and methods are providedfor at least partially neutralizing an electric charge present on afluidic droplet, for example, a fluidic droplet having an electriccharge, as described above. For example, to at least partiallyneutralize the electric charge, the fluidic droplet may be passedthrough an electric field and/or brought near an electrode, e.g., usingtechniques such as those described herein. Upon exiting of the fluidicdroplet from the electric field (i.e., such that the electric field nolonger has a strength able to substantially affect the fluidic droplet),and/or other elimination of the electric field, the fluidic droplet maybecome electrically neutralized, and/or have a reduced electric charge.

In another set of embodiments, droplets of fluid can be created from afluid surrounded by a liquid within a channel by altering the channeldimensions in a manner that is able to induce the fluid to formindividual droplets. The channel may, for example, be a channel thatexpands relative to the direction of flow, e.g., such that the fluiddoes not adhere to the channel walls and forms individual dropletsinstead, or a channel that narrows relative to the direction of flow,e.g., such that the fluid is forced to coalesce into individualdroplets. In other embodiments, internal obstructions may also be usedto cause droplet formation to occur. For instance, baffles, ridges,posts, or the like may be used to disrupt liquid flow in a manner thatcauses the fluid to coalesce into fluidic droplets.

In some cases, the channel dimensions may be altered with respect totime (for example, mechanically or electromechanically, pneumatically,etc.) in such a manner as to cause the formation of individual fluidicdroplets to occur. For example, the channel may be mechanicallycontracted (“squeezed”) to cause droplet formation, or a fluid streammay be mechanically disrupted to cause droplet formation, for example,through the use of moving baffles, rotating blades, or the like.

In some cases, an emulsion may include a larger fluidic droplet thatcontains one or more smaller droplets therein which, in some cases, cancontain even smaller droplets therein, etc. In some cases, the dropletis surrounded by a liquid (e.g., suspended). Any of these droplets maybe of substantially the same shape and/or size (i.e., “monodisperse”),or of different shapes and/or sizes, depending on the particularapplication. As used herein, the term “fluid” generally refers to asubstance that tends to flow and to conform to the outline of itscontainer, i.e., a liquid, a gas, a viscoelastic fluid, etc. Typically,fluids are materials that are unable to withstand a static shear stress,and when a shear stress is applied, the fluid experiences a continuingand permanent distortion.

The fluid may have any suitable viscosity that permits flow, forexample, a viscosity similar to water (e.g., as in an aqueous solution),oil, etc. In certain embodiments of the invention, the liquid mayinclude an oil or an organic solvent, such as those known to ordinaryskill in the art. If two or more fluids are present, each fluid may beindependently selected among essentially any fluids (liquids, gases, andthe like) by those of ordinary skill in the art, by considering therelationship between the fluids. The fluids may each be miscible orimmiscible. For example, two fluids can be selected to be immisciblewithin the time frame of formation of a stream of fluids, or within thetime frame of reaction or interaction. As an example, where the portionsremain liquid for a significant period of time, the fluids may beimmiscible. As another example, where, after contact and/or formation,the dispersed portions are quickly hardened by polymerization or thelike, the fluids need not be as immiscible. Those of ordinary skill inthe art can select suitable miscible or immiscible fluids, using contactangle measurements or the like, to carry out the techniques of theinvention.

A “droplet,” as used herein, is an isolated portion of a first fluidthat is surrounded by a second fluid. It is to be noted that a dropletis not necessarily spherical, but may assume other shapes as well, forexample, depending on the external environment. In one embodiment, thedroplet has a minimum cross-sectional dimension that is substantiallyequal to the largest dimension of the channel perpendicular to fluidflow in which the droplet is located.

As used herein, a first entity is “surrounded” by a second entity if aclosed loop can be drawn around the first entity through only the secondentity. A first entity is “completely surrounded” if closed loops goingthrough only the second entity can be drawn around the first entityregardless of direction. In one aspect, the first entity may be a cell,for example, a cell suspended in media is surrounded by the media. Inanother aspect, the first entity is a particle. In yet another aspect ofthe invention, the entities can both be fluids. For example, ahydrophilic liquid may be suspended in a hydrophobic liquid, ahydrophobic liquid may be suspended in a hydrophilic liquid, a gasbubble may be suspended in a liquid, etc. Typically, a hydrophobicliquid and a hydrophilic liquid are substantially immiscible withrespect to each other, where the hydrophilic liquid has a greateraffinity to water than does the hydrophobic liquid. Examples ofhydrophilic liquids include, but are not limited to, water and otheraqueous solutions comprising water, such as cell or biological media,ethanol, salt solutions, etc. Examples of hydrophobic liquids include,but are not limited to, oils such as hydrocarbons, silicon oils,fluorocarbon oils, organic solvents etc.

Many such oils are commercially available. As discussed above, the oilmay be chosen so as to be substantially immiscible in water, forinstance, with solubilities of less than about 50 ppb, less than about25 ppb, or less than about 10 ppb (without surfactant). Examples ofpotentially suitable hydrocarbons include, but are not limited to, lightmineral oil (Sigma), kerosene (Fluka), hexadecane (Sigma), decane(Sigma), undecane (Sigma), dodecane (Sigma), octane (Sigma), cyclohexane(Sigma), hexane (Sigma), or the like. Non-limiting examples ofpotentially suitable silicone oils include 2 cst polydimethylsiloxaneoil (Sigma). Non-limiting examples of fluorocarbon oils include FC3283(3M), FC40 (3M), Krytox GPL (Dupont), etc. In some cases, oilspotentially suitable for the invention include those that haveviscosities of between about 0.8 cSt and about 1 cSt, or between about0.7 cSt and about 0.9 cSt. In certain embodiments, the oil may have aspecific gravity of between about 1.4 and about 2, or between about 1.6and about 1.7 at 25° C., and/or a specific gravity of between about 1.2and about 1.8, or between about 1.4 and about 1.5 at 100° C. The oil mayalso have a boiling point of greater than about 100° C., or greater thanabout 120° C. in some cases. In one set of embodiments, the oil may bechosen so as to have an interfacial tension with phosphate-bufferedsaline of between about 60 mN/m and about 70 mN/m, e.g., about 63 mN/m.In some cases, a surfactant may also be present, as is discussed below.Non-limiting examples of surfactants potentially useful in the inventioninclude Span80 (Sigma), Span80/Tween-20 (Sigma), Span80/Triton X-100(Sigma), Abil EM90 (Degussa), Abil we09 (Degussa), polyglycerolpolyricinoleate “PGPR90” (Danisco), Tween-85, 749 Fluid (Dow Corning),the ammonium carboxylate salt of Krytox 157 FSL (Dupont), the ammoniumcarboxylate salt of Krytox 157 FSM (Dupont), or the ammonium carboxylatesalt of Krytox 157 FSH (Dupont).

The “cross-sectional dimension” of the channel is measured perpendicularto the direction of fluid flow. Most fluid channels in components of theinvention have maximum cross-sectional dimensions less than 2 mm, and insome cases, less than 1 mm. In one set of embodiments, all fluidchannels containing embodiments of the invention are microfluidic orhave a largest cross sectional dimension of no more than 2 mm or 1 mm.In another embodiment, the fluid channels may be formed in part by asingle component (e.g. an etched substrate or molded unit). Of course,larger channels, tubes, chambers, reservoirs, etc. can be used to storefluids in bulk and to deliver fluids to components of the invention. Inone set of embodiments, the maximum cross-sectional dimension of thechannel(s) containing embodiments of the invention are less than 500microns, less than 200 microns, less than 100 microns, less than 50microns, or less than 25 microns.

The fluidic droplets within the channels may have a cross-sectionaldimension smaller than about 90% of an average cross-sectional dimensionof the channel, and in certain embodiments, smaller than about 80%,about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about10%, about 5%, about 3%, about 1%, about 0.5%, about 0.3%, about 0.1%,about 0.05%, about 0.03%, or about 0.01% of the average cross-sectionaldimension of the channel.

In certain instances, the droplets may be contained within a carryingfluid, e.g., within a fluidic stream. The fluidic stream, in one set ofembodiments, is created using a microfluidic system, discussed in detailbelow. In some cases, the droplets will have a homogenous distributionof diameters, i.e., the droplets may have a distribution of diameterssuch that no more than about 10%, about 5%, about 3%, about 1%, about0.03%, or about 0.01% of the droplets have an average diameter greaterthan about 10%, about 5%, about 3%, about 1%, about 0.03%, or about0.01% of the average diameter of the droplets. Techniques for producingsuch a homogenous distribution of diameters are disclosed inInternational Patent Application No. PCT/US2004/010903, filed Apr. 9,2004, entitled “Formation and Control of Fluidic Species,” by Link, etal., published as WO 2004/091763 on Oct. 28, 2004, incorporated hereinby reference, and in other references as described below.

The fluidic droplets may each be substantially the same shape and/orsize. Typically, monodisperse droplets are of substantially the samesize. The shape and/or size of the fluidic droplets can be determined,for example, by measuring the average diameter or other characteristicdimension of the droplets. The “average diameter” of a plurality orseries of droplets is the arithmetic average of the average diameters ofeach of the droplets. Those of ordinary skill in the art will be able todetermine the average diameter (or other characteristic dimension) of aplurality or series of droplets, for example, using laser lightscattering, microscopic examination, or other known techniques. Theaverage diameter of a single droplet, in a non-spherical droplet, is thediameter of a perfect sphere having the same volume as the non-sphericaldroplet. The average diameter of a droplet (and/or of a plurality orseries of droplets) may be, for example, less than about 1 mm, less thanabout 500 micrometers, less than about 200 micrometers, less than about100 micrometers, less than about 75 micrometers, less than about 50micrometers, less than about 25 micrometers, less than about 10micrometers, or less than about 5 micrometers in some cases. The averagediameter may also be at least about 1 micrometer, at least about 2micrometers, at least about 3 micrometers, at least about 5 micrometers,at least about 10 micrometers, at least about 15 micrometers, or atleast about 20 micrometers in certain cases.

The term “determining,” as used herein, generally refers to the analysisor measurement of a species, for example, quantitatively orqualitatively, and/or the detection of the presence or absence of thespecies. “Determining” may also refer to the analysis or measurement ofan interaction between two or more species, for example, quantitativelyor qualitatively, or by detecting the presence or absence of theinteraction. Examples of suitable techniques include, but are notlimited to, spectroscopy such as infrared, absorption, fluorescence,UV/visible, FTIR (“Fourier Transform Infrared Spectroscopy”), or Raman;gravimetric techniques; ellipsometry; piezoelectric measurements;immunoassays; electrochemical measurements; optical measurements such asoptical density measurements; circular dichroism; light scatteringmeasurements such as quasielectric light scattering; polarimetry;refractometry; or turbidity measurements.

In some cases, a plurality of droplets may be substantially the same. Itshould be understood that, even if the droplets appear to besubstantially identical, or to contain substantially the same number ofdroplets therein, not all of the droplets will necessarily be completelyidentical. In some cases, there may be minor variations in the numberand/or size of droplets contained within a surrounding droplet. Thus, insome cases, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 92%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% of a plurality of outer droplets may each containthe same number of inner fluidic droplets therein.

As used herein, the term “fluid stream” or “fluidic stream” refers tothe flow of a fluid, typically generally in a specific direction. Thefluidic stream may be continuous and/or discontinuous. A “continuous”fluidic stream is a fluidic stream that is produced as a single entity,e.g., if a continuous fluidic stream is produced from a channel, thefluidic stream, after production, appears to be contiguous with thechannel outlet. The continuous fluidic stream may be laminar, orturbulent in some cases. The continuous fluidic stream may be, e.g.,solid or hollow (i.e., containing a second fluid internally, forexample, as in a hollow tube). It is to be understood that wherever“tube” is used herein, the structure can be a hollow, a solid or filled(i.e., not hollow) stream, a stream that includes a central core and asurrounding layer or layers, any of which can be selectively reactedwith any others, or solidified, or the like. In some cases, the centralcore is hollow, and/or fluid may be removed from a hardened surroundingfluid to produce a hollow tube.

Similarly, a “discontinuous” fluidic stream is a fluidic stream that isnot produced as a single entity. A discontinuous fluidic stream may havethe appearance of individual droplets, optionally surrounded by a secondfluid. A “droplet,” as used herein, is an isolated portion of a firstfluid that completely surrounded by a second fluid. In some cases, thedroplets may be spherical or substantially spherical; however, in othercases, the droplets may be non-spherical, for example, the droplets mayhave the appearance of “blobs” or other irregular shapes, for instance,depending on the external environment.

In another aspect, the methods and apparatus of the invention can beused to form droplets containing species and to provide methods ofdelivering such species. For example, in certain embodiments of theinvention, the fluidic droplets may contain additional entities orspecies, for example, other chemical, biochemical, or biologicalentities (e.g., dissolved or suspended in the fluid), cells, particles,beads, gases, molecules, pharmaceutical agents, drugs, DNA, RNA,proteins, fragrance, reactive agents, biocides, fungicides,preservatives, chemicals, or the like. Cells, for example, can besuspended in a fluid multiple emulsion, or contained in a polymerosome.Thus, the species may be any substance that can be contained in anyportion of a droplet and can be differentiated from the droplet fluid.The species may be present in any fluidic droplet, for example, withinan inner droplet and/or within an outer droplet, etc. In some cases, thedroplets may each be substantially the same shape or size, as discussedabove. In certain embodiments, any of the droplets disclosed herein(e.g., a hardened droplet) may contain one or more species.

As the polydispersity and size of the droplets can be narrowlycontrolled, emulsions can be formed that include a specific number ofspecies or particles per droplet. For instance, a single droplet maycontain 1, 2, 3, 4, or more species. The emulsions can be formed withlow polydispersity so that greater than 90%, 95%, or 99% of the dropletsformed contain the same number of species. In certain instances, theinvention provides for the production of droplets consisting essentiallyof a substantially uniform number of entities of a species therein(i.e., molecules, cells, particles, etc.). For example, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 92%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%, ormore of a plurality or series of droplets may each contain at least oneentity, and/or may contain the same number of entities of a particularspecies. For instance, a substantial number of fluidic dropletsproduced, e.g., as described above, may each contain 1 entity, 2entities, 3 entities, 4 entities, 5 entities, 7 entities, 10 entities,15 entities, 20 entities, 25 entities, 30 entities, 40 entities, 50entities, 60 entities, 70 entities, 80 entities, 90 entities, 100entities, etc., where the entities are molecules or macromolecules,cells, particles, etc. In some cases, the droplets may eachindependently contain a range of entities, for example, less than 20entities, less than 15 entities, less than 10 entities, less than 7entities, less than 5 entities, or less than 3 entities in some cases.

In one set of embodiments, in a plurality of droplets of fluid, some ofwhich contain a species of interest and some of which do not contain thespecies of interest, the droplets of fluid may be screened or sorted forthose droplets of fluid containing the species, and in some cases, thedroplets may be screened or sorted for those droplets of fluidcontaining a particular number or range of entities of the species ofinterest. Systems and methods for screening and/or sorting droplets aredisclosed in, for example, U.S. patent application Ser. No. 11/360,845,filed Feb. 23, 2006, entitled “Electronic Control of Fluidic Species,”by Link, et al., published as U.S. Patent Application Publication No.2007/000342 on Jan. 4, 2007, incorporated herein by reference.

Thus, in some cases, a plurality or series of fluidic droplets, some ofwhich contain the species and some of which do not, may be enriched (ordepleted) in the ratio of droplets that do contain the species, forexample, by a factor of at least about 2, at least about 3, at leastabout 5, at least about 10, at least about 15, at least about 20, atleast about 50, at least about 100, at least about 125, at least about150, at least about 200, at least about 250, at least about 500, atleast about 750, at least about 1000, at least about 2000, or at leastabout 5000 or more in some cases. In other cases, the enrichment (ordepletion) may be in a ratio of at least about 10⁴, at least about 10⁵,at least about 10⁶, at least about 10⁷, at least about 10⁸, at leastabout 10⁹, at least about 10¹⁰, at least about 10¹¹, at least about10¹², at least about 10¹³, at least about 10¹⁴, at least about 10¹⁵, ormore. For example, a fluidic droplet containing a particular species maybe selected from a library of fluidic droplets containing variousspecies, where the library may have about 10⁵, about 10⁶, about 10⁷,about 10⁸, about 10⁹, about 10¹⁰, about 10¹¹, about 10¹², about 10¹³,about 10¹⁴, about 10¹⁵, or more items, for example, a DNA library, anRNA library, a protein library, a combinatorial chemistry library, etc.In certain embodiments, the droplets carrying the species may then befused, reacted, or otherwise used or processed, etc., as furtherdescribed herein, for example, to initiate or determine a reaction.

In one set of embodiments, the fluidic droplets may contain cells orother entities, such as proteins, viruses, macromolecules, particles,etc. As used herein, a “cell” is given its ordinary meaning as used inbiology. One or more cells and/or one or more cell types can becontained in a droplet. The inner fluid may be, for example, an aqueousbuffer solution. The cell may be any cell or cell type. For example, thecell may be a bacterium or other single-cell organism, a plant cell, oran animal cell. If the cell is a single-cell organism, then the cell maybe, for example, a protozoan, a trypanosome, an amoeba, a yeast cell,algae, etc. If the cell is an animal cell, the cell may be, for example,an invertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g.,a zebrafish cell), an amphibian cell (e.g., a frog cell), a reptilecell, a bird cell, or a mammalian cell such as a primate cell, a bovinecell, a horse cell, a porcine cell, a goat cell, a dog cell, a cat cell,or a cell from a rodent such as a rat or a mouse. If the cell is from amulticellular organism, the cell may be from any part of the organism.For instance, if the cell is from an animal, the cell may be a cardiaccell, a fibroblast, a keratinocyte, a heptaocyte, a chondracyte, aneural cell, a osteocyte, a muscle cell, a blood cell, an endothelialcell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, aneutrophil, a basophil, a mast cell, an eosinophil), a stem cell, etc.In some cases, the cell may be a genetically engineered cell. In certainembodiments, the cell may be a Chinese hamster ovarian (“CHO”) cell or a3T3 cell.

For example, an emulsion can be formed in which greater than about 95%of the droplets formed contain a single cell at the point of dropletproduction, without a need to separate or otherwise purify the emulsionin order to obtain this level of dispersity. Typically, the fluidsupporting the cell is the innermost fluid and is aqueous based. Thesurrounding fluid may be a non-aqueous fluid and other fluids, e.g.,within a multiple emulsion, may be aqueous or non-aqueous. If apolymerosome is used, the shell surrounding the cell (which may or maynot be the outermost fluidic droplet in a multiple emulsion) may beformed of a material capable of protecting the cell. The shell may helpretain, for example, moisture, and can be sized appropriately tomaximize the lifetime of the cell within the polymerosome. For instance,the shell may be sized to contain a specific volume, e.g., 10 mL, ofinner fluid as well as a single cell or a select number of cells.Likewise, cells may be suspended so that, statistically, one cell willbe included with each aliquot (e.g., 10 mL) of fluid within a droplet.

In one set of embodiments, a fluidic droplet of the present invention,or a portion thereof, may be hardened into a solid. As used herein, the“hardening” of a fluidic stream refers to a process by which at least aportion of the fluidic stream is converted into a solid or at least asemi-solid state (e.g., a gel, a viscoelastic solid, etc.). In someembodiments of the invention, a droplet may be hardened, such as byusing a fluid that can be solidified, gelled, and/or polymerized (e.g.,to form a polymerosome). The droplet may be an outer droplet or onecontained within a surrounding droplet. In some cases, capsules orspheres can be formed, i.e., by hardening a droplet containing one ormore fluidic droplets therein. For example, a solid sphere may be formedif an inner droplet is hardened, e.g., to form a gel. Any technique ableto solidify a fluidic droplet can be used. For example, a fluidicdroplet may be cooled to a temperature below the melting point or glasstransition temperature of a fluid within the fluidic droplet, a chemicalreaction may be induced that causes the fluidic droplet to solidify (forexample, a polymerization reaction, a reaction between two fluids thatproduces a solid product, etc.), or the like. In some cases, thehardened droplet may contain an entity or species, as described above.For example, a droplet containing a cell may be hardened to form a gel.As another example, a droplet of an outer fluid, containing an innerfluid containing a cell, may be hardened to produce a gel “capsule”surrounding the inner fluid and the cell. In some cases, a probiotic maybe incorporated or encapsulated within a gel or other hardened particleas a way to increase stability.

In some embodiments of the invention, a hardened shell may be formedaround an inner droplet, such as by using a middle fluid that can besolidified or gelled. In this way, capsules can be formed withconsistently and repeatedly-sized inner droplets, as well as aconsistent and repeatedly-sized outer shell. In some embodiments, thiscan be accomplished by a phase change in the middle fluid.

In one embodiment, the fluidic droplet is solidified by reducing thetemperature of the fluidic droplet to a temperature that causes at leastone of the components of the fluidic droplet to reach a solid state. Forexample, the fluidic droplet may be solidified by cooling the fluidicdroplet to a temperature that is below the melting point or glasstransition temperature of a component of the fluidic droplet, therebycausing the fluidic droplet to become solid. As non-limiting examples,the fluidic droplet may be formed at an elevated temperature (i.e.,above room temperature, about 25° C.), then cooled, e.g., to roomtemperature or to a temperature below room temperature; the fluidicdroplet may be formed at room temperature, then cooled to a temperaturebelow room temperature, or the like. As a specific example, a fluidicdroplet may contain a gel such as a hydrogel, and the droplet may besolidified by cooling the droplet below its gelation temperature.

In some embodiments, this can be accomplished by a phase change in afluid forming the droplet. A phase change can be initiated by atemperature change, for instance, and in some cases the phase change isreversible. For example, a wax or gel may be used as a fluid at atemperature which maintains the wax or gel as a fluid. Upon cooling, thewax or gel can form a solid or semisolid shell, e.g., resulting in acapsule or a hardened particle. In another embodiment, hardening can beaccomplished by polymerizing a fluid. This can be accomplished in anumber of ways, including using a pre-polymer that can be catalyzed, forexample, chemically, through heat, or via electromagnetic radiation(e.g., ultraviolet radiation) to form a solid polymer shell or particle.

Non-limiting examples of gel systems that can be used in the presentinvention include acrylamide-based gels, such as polyacrylamide or polyN-isopropylpolyacrylamide. For example, an aqueous solution of a monomermay be dispersed in a droplet, and then polymerized, e.g., to form agel. Another example is a hydrogel, such as alginic acid, that can begelled by the addition of calcium ions. In some cases, gelationinitiators (ammonium persulfate and TEMED for acrylamide, or Ca²⁺ foralginate) can be added to a droplet, for example, by co-flow with theaqueous phase, by co-flow through the oil phase, or by coalescence oftwo different drops, e.g., as discussed in detail below, or as disclosedin U.S. patent application Ser. No. 11/360,845, filed Feb. 23, 2006,entitled “Electronic Control of Fluidic Species,” by Link, et al.,published as U.S. Patent Application Publication No. 2007/000342 on Jan.4, 2007; or in U.S. patent application Ser. No. 11/698,298, filed Jan.24, 2007, entitled “Fluidic Droplet Coalescence,” by Ahn, et. al; eachincorporated herein by reference.

As a specific non-limiting example, in one embodiment, a dropletcontaining a cell is hardened, e.g., forming a gel particle containingthe cell, or a gel capsule surrounding the cell, etc. If a gelformulation (or other hardening system) is used that is not toxic tocells, such as alginate, the cells may be grown within the gel matrix.The gel is a solid substrate on which to grow, and if a single cell ispresent in a gel at the time of formation, then any additional cellswill be daughter cells of the original, and therefore the microcolony ofcells in the gel may be formed, which may be a clonal population. Insome cases, the cells within the gel would be “cloaked” from the immunesystem if injected into a subject, i.e., at least some of the immunesystem components from the subject are unable to penetrate the gel todirectly access the cell.

In one aspect, after formation of an emulsion, one or more fluids may beremoved. For example, droplets may be separated from a carrying fluid,or an inner droplet may be separated from an outer droplet. As aspecific non-limiting example, in cases where it may be desirable toremove a portion of the middle fluid from the outer drop, for example,when forming a shell through self-assembly, some of the components ofthe middle fluid may be at least partially miscible in the outer fluid.This can allow the components to diffuse over time into the outersolvent, reducing the concentration of the components in the outerdroplet, which can effectively increase the concentration of any of theimmiscible components, e.g., polymers or surfactants, that comprise theouter droplet. This can lead to the self-assembly or gelation ofpolymers or other shell precursors in some embodiments, and can resultin the formation of a solid or semi-solid shell. During dropletformation, it may still be desired that the middle fluid be at leastsubstantially immiscible with the outer fluid. This immiscibility can beprovided, for example, by polymers, surfactants, solvents, or othercomponents that form a portion of the middle fluid, but are not able toreadily diffuse, at least entirely, into the outer fluid after dropletformation. Thus, the middle fluid can include, in certain embodiments,both a miscible component that can diffuse into the outer fluid afterdroplet formation, and an immiscible component that helps to promotedroplet formation.

As another example, in one set of embodiments, a droplet, which may behardened droplet, may be removed from a fluid carrying the droplet, andin some cases, the droplet may be placed in a third fluid. As a specificnon-limiting example, an aqueous droplet suspended in an oil-based fluidmay be removed from the oil-based fluid by causing hardening of theaqueous droplet, e.g., forming a gel, then the carrying or surroundingfluid may be removed, e.g., by washing away with a suitable solvent.Optionally, the droplet may be placed in an aqueous solution.

In another set of embodiments, fluid can be removed from an innerdroplet in order to, for example, concentrate any species that may becontained within the inner droplet. Fluid may be removed from the innerdroplet, or the inner droplet may be concentrated, using techniquessimilar to those described herein for removing fluid from an outerdroplet. For instance, fluid can diffuse from or evaporate out of theinner droplet in order to reduce the size of the inner droplet, andtherefore concentrate any components of the inner droplet that do notsubstantially diffuse or evaporate. For example, the volume of an innerdroplet can be reduced by more than 50%, 75%, 90%, 95%, 99%, or 99.9%.Thus, the core radius of the inner droplet can be reduced by, forexample, a factor of 2, 5, 10, or more, in some cases.

Fluid components can be chosen by those skilled in the art forparticular diffusion or evaporative characteristics. The middle fluid(outer droplet) can also be selected so that the middle fluid providesfor transfer of the inner fluid, either into or through the middlefluid. The size (thickness) of the outer droplet may also affect therate of transfer out of the inner droplet, and in some cases thethickness of the outer droplet can be selected in order to control therate at which inner fluid is removed from the inner droplet. Those ofordinary skill in the art will be able to optimize such a system, usingno more than routine skill, to achieve a desired diffusion or evaporatea characteristic, depending on the particular application.

According to still another set of embodiments, a specific shell materialmay be chosen to dissolve, rupture, or otherwise release its contentsunder certain conditions. For example, if a polymerosome contains adrug, the shell components may be chosen to dissolve under certainphysiological conditions (e.g., pH, temperature, osmotic strength),allowing the drug to be selectively released. Materials useful in these“smart capsules” are known to those skilled in the art. If it is desiredthat the inner species be dried, the shell material may be of asubstance that is permeable to water molecules.

Any of the fluids within an emulsion droplet may the same, or different.For example, the fluids may be chosen such that the inner dropletsremain discrete, relative to their surroundings. As non-limitingexamples, a fluidic droplet may be created having an outer droplet,containing one or more first fluidic droplets, some or all of which maycontain one or more second fluidic droplets. In some cases, the outerfluid and the second fluid may be identical or substantially identical;however, in other cases, the outer fluid, the first fluid, and thesecond fluid may be chosen to be essentially mutually immiscible. Onenon-limiting example of a system involving three essentially mutuallyimmiscible fluids is a silicone oil, a mineral oil, and an aqueoussolution (i.e., water, or water containing one or more other speciesthat are dissolved and/or suspended therein, for example, a saltsolution, a saline solution, a suspension of water containing particlesor cells, or the like). Another example of a system is a silicone oil, afluorocarbon oil, and an aqueous solution. Yet another example of asystem is a hydrocarbon oil (e.g., hexadecane), a fluorocarbon oil, andan aqueous solution. Non-limiting examples of suitable fluorocarbon oilsinclude octadecafluorodecahydronaphthalene:

or 1-(1,2,2,3,3,4,4,5,5,6,6-undecafluorocyclohexyl)ethanol:

In the descriptions herein, multiple emulsions are often described withreference to a three phase system, i.e., having a carrying fluid, anouter fluid, and an inner fluid. However, it should be noted that thisis by way of example only, and that in other systems, additional fluidsor fewer fluids may be present within the multiple emulsion droplet.Accordingly, it should be understood that the descriptions of thecarrying fluid, outer fluid, and inner fluid are by way of ease ofpresentation, and that the descriptions herein are readily extendable tosystems involving additional fluids, e.g., quadruple emulsions,quintuple emulsions, sextuple emulsions, septuple emulsions, etc.

As a non-limiting example, in one set of embodiments, a triple emulsionmay be produced, i.e., an emulsion containing outer fluid, containingdroplets containing an outer fluid, some of which droplets can containone or more inner fluidic droplets therein. In some cases, the carryingfluid and the inner fluid may be the same. The fluids in the tripleemulsion are often of varying miscibilities, due to differences inhydrophobicity. For example, the carrying fluid may be water soluble(i.e., miscible in water), the outer fluid oil soluble (or immiscible inwater), and the inner fluid water soluble. This arrangement is oftenreferred to as a w/o/w multiple emulsion (“water/oil/water”). Anothermultiple emulsion may include a carrying fluid that is oil soluble (orimmiscible in water), an outer fluid that is water soluble, and an innerfluid that is oil soluble. This type of multiple emulsion is oftenreferred to as an o/w/o multiple emulsion (“oil/water/oil”). It shouldbe noted that the term “oil” in the above terminology merely refers to afluid that is generally more hydrophobic and not miscible in water, asis known in the art. Thus, the oil may be a hydrocarbon in someembodiments, but in other embodiments, the oil may comprise otherhydrophobic fluids.

More specifically, as used herein, two fluids are immiscible, or notmiscible, with each other when one is not soluble in the other to alevel of at least 10% by weight at the temperature and under theconditions at which the multiple emulsion is produced. For instance, twofluids may be selected to be immiscible within the time frame of theformation of the fluidic droplets. In some embodiments, the carrying andinner fluids are compatible, or miscible, while the outer fluid isincompatible or immiscible with one or both of the carrying and innerfluids. In other embodiments, however, all three fluids may be mutuallyimmiscible, and in certain cases, all of the fluids do not allnecessarily have to be water soluble.

As fluid viscosity can affect droplet formation, in some cases theviscosity of any of the fluids in the fluidic droplets may be adjustedby adding or removing components, such as diluents, that can aid inadjusting viscosity. For example, in some embodiments, the viscosity ofthe outer fluid and the first fluid are equal or substantially equal.This may aid in, for example, an equivalent frequency or rate of dropletformation in the outer and fluid fluids. In other embodiments, theviscosity of the first fluid may be equal or substantially equal to theviscosity of the second fluid, and/or the viscosity of the outer fluidmay be equal or substantially equal to the viscosity of the secondfluid. In yet another embodiment, the outer fluid may exhibit aviscosity that is substantially different from either the first orsecond fluids. A substantial difference in viscosity means that thedifference in viscosity between the two fluids can be measured on astatistically significant basis. Other distributions of fluidviscosities within the droplets are also possible. For example, thesecond fluid may have a viscosity greater than or less than theviscosity of the first fluid (i.e., the viscosities of the two fluidsmay be substantially different), the first fluid may have a viscositythat is greater than or less than the viscosity of the outer fluid, etc.It should also be noted that, in higher-order droplets, e.g., containingfour, five, six, or more fluids, the viscosities may also beindependently selected as desired, depending on the particularapplication.

In one aspect of the invention, multiple emulsions can be formed thatinclude amphiphilic species such as amphiphilic polymers and lipids andamphiphilic species typically includes a relatively hydrophilic portion,and a relatively hydrophobic portion. For instance, the hydrophilicportion may be a portion of the molecule that is charged, and thehydrophobic portion of the molecule may be a portion of the moleculethat comprises hydrocarbon chains. The polymerosomes may be formed, forexample, in devices such as those described above with respect tomultiple emulsions. As mentioned above, one or more of the fluidsforming the multiple emulsions may include polymers, such as copolymers,which can be subsequently polymerized. An example of such a system isnormal butyl acrylate and acrylic acid, which can be polymerized to forma copolymer of poly(normal-butyl acrylate)-poly(acrylic acid).

Other amphiphilic species may also be used, besides diblock copolymers.For example, other polymers, or other species such as lipids orphospholipids may be used with the present invention. For example,liposomes can also be formed from phospholipids and/or other lipids. Forexample, lipids or phospholipids may be provided instead of polymers inthe methods described above. Other methods may also be used to producerobust encapsulants, for example, surface-induced polymerization ofeither the inner or outer interface, or temperature-induced gelation ofthe inner or middle fluid.

When lipids are used, the resulting emulsion droplets are typicallyreferred to as vesicles or lipid vesicles. When an amphiphilic polymer,such as a diblock copolymer, is used, the resulting droplets can bereferred to as polymerosomes. “Polymers,” as used herein, may includepolymeric compounds, as well as compounds and species that can formpolymeric compounds, such as prepolymers. Prepolymers include, forexample, monomers and oligomers. In some cases, however, only polymericcompounds are used and prepolymers may not be appropriate.

Upon formation of a multiple emulsion, an amphiphilic species that iscontained, dissolved, or suspended in the emulsion can spontaneouslyassociate along a hydrophilic/hydrophobic interface in some cases. Forinstance, the hydrophilic portion of an amphiphilic species may extendinto the aqueous phase and the hydrophobic portion may extend into thenon-aqueous phase. Thus, the amphiphilic species can spontaneouslyorganize under certain conditions so that the amphiphilic speciesmolecules orient substantially parallel to each other and are orientedsubstantially perpendicular to the interface between two adjoiningfluids, such as an inner droplet and outer droplet, or an outer dropletand an outer fluid. As the amphiphilic species become organized, theymay form a sheet, e.g., a substantially spherical sheet, with ahydrophobic surface and an opposed hydrophilic surface. Depending on thearrangement of fluids, the hydrophobic side may face inwardly oroutwardly and the hydrophilic side may face inwardly or outwardly. Theresulting multiple emulsion structure may be a bilayer or amulti-lamellar structure.

In one set of embodiments, a method of forming multiple emulsionstructures containing amphiphilic species, such as polymer vesicles or“polymerosomes,” involves the removal of a portion of the middle fluidafter the formation of a multiple emulsion. For instance, a component ofthe middle fluid, such as a solvent or carrier, can be removed from thefluid, in part or in whole, through evaporation or diffusion. Theremaining component or components of the middle fluid may self-organizeor otherwise harden as a result of the reduction in the amount ofsolvent or carrier in the middle fluid, similar to those processespreviously described. This shell formation can occur, for example,through crystallization or self-assembly of polymers dissolved in themiddle fluid. For instance, a surfactant or surfactants can be used sothat when the surfactant concentration in the middle fluid increases(e.g., concurrently with a decrease in the solvent concentration) thesurfactant molecules are oriented so that like regions of the surfactantare associated with the inner droplet and/or the outer fluid. Within theshell itself (i.e., the middle fluid), different regions of thesurfactant molecules may associate with each other, resulting in aconcentrating of materials that then form a membrane of lamellarsheet(s) composed primarily or substantially of surfactant. The membranemay be solid or semi-solid in some cases. Non-surfactants can also beused.

In some cases, the middle fluid comprises a solvent system used as acarrier, and a dissolved or suspended polymer such as a diblockcopolymer, which can be amphiphilic. After formation of a multipleemulsion, the solvent can be removed from the shell using techniquessuch as evaporation or diffusion, leaving the diblock copolymers behind.As the solvent leaves the middle fluid layer, the polymers canself-assemble into single or multiple layers on the inner and/or outersurfaces, resulting in a polymerosome. This can result in a thinmembrane that is capable of carrying, protecting, and delivering theinner droplet. Once formed, these polymerosomes can be removed from theouter fluid, dried, stored, etc.

In one set of embodiments, one or more fluids within the multipleemulsion may be polymerized, e.g., to form a polymerosome. For instance,in some cases, one or more of the fluids forming the multiple emulsionsmay include polymers, such as copolymers, which can be subsequentlypolymerized. An example of such a system is normal butyl acrylate andacrylic acid, which can be polymerized to form a copolymer ofpoly(normal-butyl acrylate)-poly(acrylic acid).

The schematic diagram illustrated in FIG. 5 shows one embodiment of theinvention including a device 100 having an outer conduit 110, a firstinner conduit (or injection tube) 120, and a second inner conduit (orcollection tube) 130. An inner fluid 140 is shown flowing in a right toleft direction and middle fluid 150 flows in a right to left directionin the space outside of injection tube 120 and within conduit 110. Outerfluid 160 flows in a left to right direction in the pathway providedbetween outer conduit 110 and collection tube 130. After outer fluid 160contacts middle fluid 150, it changes direction and starts to flow insubstantially the same direction as the inner fluid 140 and the middlefluid 150, right to left. Injection tube 120 includes an exit orifice164 at the end of tapered portion 170. Collection tube 130 includes anentrance orifice 162, an internally tapered surface 172, and exitchannel 168. Thus, the inner diameter of injection tube 120 decreases ina direction from right to left, as shown, and the inner diameter ofcollection tube 130 increases from the entrance orifice in a directionfrom right to left. These constrictions, or tapers, can providegeometries that aid in producing consistent emulsions. The rate ofconstriction may be linear or non-linear.

Still referring to the example shown in FIG. 5, inner fluid 140 exitingfrom orifice 164 can be completely surrounded by middle fluid 150, asthere is no portion of inner fluid 140 that contacts the inner surfaceof conduit 110 after its exit from injection tube 120. Thus, for aportion between exit orifice 164 to a point inside of collection tube130 (to the left of entrance orifice 162), a stream of fluid 140 isconcentrically surrounded by a stream of fluid 150. Additionally, middlefluid 150 may not come into contact with the surface of collection tube130, at least until after the emulsion has been formed, because it isconcentrically surrounded by outer fluid 160 as it enters collectiontube 130. Thus, from a point to the left of exit orifice 164 to a pointinside of collection tube 130, a composite stream of three fluid streamsis formed, including inner fluid 140 concentrically surrounded by astream of middle fluid 150, which in turn is concentrically surroundedby a stream of outer fluid 160. The inner and middle fluids do nottypically break into droplets until they are inside of collection tube130 (to the left of entrance orifice 162). Under “dripping” conditions,the droplets are formed closer to the orifice, while under “jetting”conditions, the droplets are formed further downstream, i.e., to theleft.

Dripping conditions produce droplets close to the entrance of collectiontube 130 (FIG. 5) within a single orifice diameter; this can beanalogized to a dripping faucet. Droplets produced by dripping aretypically substantially monodisperse. By contrast, jetting conditionsproduce a long jet that extends three or more orifice diametersdownstream into the collection tube, where it breaks into droplets.Although the distance from the opening may be greater under the jettingregime, droplets formed by either method are typically formed inside thecollection tube. The jetting regime is typically quite irregular,resulting in polydisperse droplets, whose radius is much greater thanthat of the jet. Jet formation is believed to be caused by the viscousstress of the outer fluid on the middle fluid. When viscous effectsdominate over inertial effects, the Reynolds number is low. Theformation of multiple emulsions is similar to that of single emulsions;however, there are at least two fluids flowing coaxially, each of whichcan form droplets through either mechanism.

Droplet formation and morphology can be affected in a number of ways.For example, the geometry of the device, including the relationship ofan outer conduit and two inner conduits, can be useful in developingmultiple emulsions of desired size, frequency, and content. For example,the size of the orifice 162 and the inner taper of collection tube 130can help to maintain three fluids in position, allowing droplets 180 toform. In addition, droplet formation can be affected by the rate of flowof the inner fluid, the rate of flow of the middle fluid, the rate offlow of the outer fluid, the total amount of flow or a change in theratios, and/or combinations of any of these flow rates. In someembodiments, multiple droplets of inner fluid can be formed within asingle droplet of the middle fluid. For example, 2, 3, 4, 5, 10, 30,100, 300, 1000 or more droplets of inner fluid can be formed within adroplet of middle fluid by varying the frequency of droplet formation ofeither (or both) the inner fluid or the middle fluid, in relation to theother of the inner fluid or the middle fluid. For example, if thevelocity of the inner fluid is altered so that five droplets are formedover the same amount of time as a single droplet of middle fluid, then adroplet of middle fluid may contain, on average, five droplets of innerfluid. It should be noted that, depending on the fluid flowcharacteristics, some of the middle fluid droplets may contain more orfewer droplets of inner fluid, although the average is five droplets, asdiscussed in this example. As the absolute and relative flow rates ofthe three fluids can be carefully controlled using the devices describedherein, the middle fluid droplets containing specific numbers of innerfluid droplets can be consistently and repeatedly formed. In someembodiments, the standard deviation from a target number of inner fluiddroplets per middle fluid droplet may be, for example, less than oneinner droplet, or less than about 20% of the number of inner dropletsper middle fluid droplet. In other embodiments, the standard deviationmay be, for example, less than about 15%, less than about 12%, less thanabout 10%, less than about 8%, or less than about 6% of the number ofinner droplets per middle fluid droplet. In some cases, substantiallyall of the outer droplets will contain the same number of dropletstherein.

The relative sizes of the inner fluid droplet and the middle fluiddroplet can also be carefully controlled, i.e., the ratio of the size ofthe inner and outer droplets can be predicatively controlled. Forinstance, inner fluid droplets may fill much of or only a small portionof the middle fluid (outer) droplet. Inner fluid droplets may fill lessthan about 90%, less than about 80%, less than about 70%, less thanabout 60%, less than about 50%, less than about 30%, less than about20%, or less than about 10% of the volume of the outer droplet.Alternatively, the inner fluid droplet may form greater than about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about90%, about 95%, or about 99% of the volume of the outer droplet. In somecases, the outer droplet can be considered a fluid shell, or coating,when it contains an inner droplet, as some or most of the outer dropletvolume may be filled by the inner droplet. The ratio of the middle fluidshell thickness to the middle fluid droplet radius can be equal to orless than, e.g., about 5%, about 4%, about 3%, or about 2%. This canallow, in some embodiments, for the formation of multiple emulsions withonly a very thin layer of material separating, and thus stabilizing, twomiscible fluids. The middle shell material can also be thickened togreater than or equal to, e.g., about 10%, about 20%, about 30%, about40%, or about 50% of the middle fluid droplet radius.

In some cases, such as when droplets of middle fluid 150 (outerdroplets) are formed at the same rate as are droplets of inner fluid140, then there is a one-to-one correspondence between inner fluid andmiddle fluid droplets, and each droplet of inner fluid is surrounded bya droplet of middle fluid, and each droplet of middle fluid contains asingle inner droplet of inner fluid. The term “outer droplet,” in thiscase, means a fluid droplet containing an inner fluid droplet thatcomprises a different fluid. In many embodiments that use three fluidsfor multiple emulsion production, the outer droplet is formed from amiddle fluid and not from the outer fluid as the term may imply. Itshould be noted that the above-described figure and description is byway of example only, and other multiple emulsions (having differingnumbers of nesting levels), and other devices are also contemplatedwithin the instant invention. For example, the device in FIG. 5 may bemodified to include additional concentric tubes, for example, to producemore highly nested droplets. Even higher degrees of nesting arepossible, for example, four concentric tubes, five concentric tubes, orthe like. It should be noted that “concentric,” as used herein, does notnecessarily refer to tubes that strictly coaxial, but also includesnested or “off-center” tubes that do not share a common center line.

The rate of production of droplets may be determined by the dropletformation frequency, which under many conditions can vary betweenapproximately 100 Hz and 5000 Hz. In some cases, the rate of dropletproduction may be at least about 200 Hz, at least about 300 Hz, at leastabout 500 Hz, at least about 750 Hz, at least about 1,000 Hz, at leastabout 2,000 Hz, at least about 3,000 Hz, at least about 4,000 Hz, or atleast about 5,000 Hz.

Production of large quantities of multiple emulsions can be facilitatedby the parallel use of multiple devices such as those described herein,in some instances. In some cases, relatively large numbers of devicesmay be used in parallel, for example at least about 10 devices, at leastabout 30 devices, at least about 50 devices, at least about 75 devices,at least about 100 devices, at least about 200 devices, at least about300 devices, at least about 500 devices, at least about 750 devices, orat least about 1,000 devices or more may be operated in parallel. Thedevices may comprise different conduits (e.g., concentric conduits),orifices, microfluidics, etc. In some cases, an array of such devicesmay be formed by stacking the devices horizontally and/or vertically.The devices may be commonly controlled, or separately controlled, andcan be provided with common or separate sources of various fluids,depending on the application.

The invention, in yet another aspect, relates to systems and methods forfusing or coalescing two or more fluidic droplets into one droplet. Forexample, in one set of embodiments, systems and methods are providedthat are able to cause two or more droplets (e.g., arising fromdiscontinuous streams of fluid) to fuse or coalesce into one droplet incases where the two or more droplets ordinarily are unable to fuse orcoalesce, for example, due to composition, surface tension, dropletsize, the presence or absence of surfactants, etc. In certainmicrofluidic systems, the surface tension of the droplets, relative tothe size of the droplets, may also prevent fusion or coalescence of thedroplets from occurring in some cases.

In one embodiment, two fluidic droplets may be given opposite electriccharges (i.e., positive and negative charges, not necessarily of thesame magnitude), which may increase the electrical interaction of thetwo droplets such that fusion or coalescence of the droplets can occurdue to their opposite electric charges, e.g., using the techniquesdescribed herein. For instance, an electric field may be applied to thedroplets, the droplets may be passed through a capacitor, a chemicalreaction may cause the droplets to become charged, etc. The droplets, insome cases, may not be able to fuse even if a surfactant is applied tolower the surface tension of the droplets. However, if the fluidicdroplets are electrically charged with opposite charges (which can be,but are not necessarily of, the same magnitude), the droplets may beable to fuse or coalesce.

In another embodiment, the fluidic droplets may not necessarily be givenopposite electric charges (and, in some cases, may not be given anyelectric charge), and are fused through the use of dipoles induced inthe fluidic droplets that causes the fluidic droplets to coalesce.

It should be noted that, in various embodiments, the two or moredroplets allowed to coalesce are not necessarily required to meet“head-on.” Any angle of contact, so long as at least some fusion of thedroplets initially occurs, is sufficient.

Other examples of fusing or coalescing fluidic droplets are described inInternational Patent Application Serial No. PCT/US2004/010903, filedApr. 9, 2004 by Link, et al., incorporated herein by reference.

In embodiments where an electric field may be applied to two (or more)fluidic droplets to cause the droplets to fuse or coalesce, theelectrical charge may be created using any suitable techniques known tothose of ordinary skill in the art; for example, an electric field maybe imposed on a channel containing the droplets, the droplets may bepassed through a capacitor, a chemical reaction may occur to cause thedroplets to become charged, etc. For instance, in one embodiment, anelectric field may be generated proximate a portion of a channel, suchas a microfluidic channel. The electric field may be generated from, forexample, an electric field generator, i.e., a system able to produce anelectric field, e.g., directed substantially at the channel. Techniquesfor producing a suitable electric field are known to those of ordinaryskill in the art. For example, an electric field may be produced byapplying a voltage across electrodes positioned proximate a channel,e.g., as shown in FIG. 3B. The electrodes can be fashioned from anysuitable electrode material, for example, as silver, gold, copper,carbon, platinum, copper, tungsten, tin, cadmium, nickel, indium tinoxide (“ITO”), etc., as is known to those of ordinary skill in the art.The electrodes may be formed of the same material, or differentmaterials. In some cases, transparent or substantially transparentelectrodes may be used.

In certain embodiments, the electric field generator may be constructedand arranged to generate an electric field within a fluid of at leastabout 0.01 V/micrometer, and, in some cases, at least about 0.03V/micrometer, at least about 0.05 V/micrometer, at least about 0.08V/micrometer, at least about 0.1 V/micrometer, at least about 0.3V/micrometer, at least about 0.5 V/micrometer, at least about 0.7V/micrometer, at least about 1 V/micrometer, at least about 1.2V/micrometer, at least about 1.4 V/micrometer, at least about 1.6V/micrometer, or at least about 2 V/micrometer. In some embodiments,even higher electric fields may be used, for example, at least about 2V/micrometer, at least about 3 V/micrometer, at least about 5V/micrometer, at least about 7 V/micrometer, or at least about 10V/micrometer or more.

The applied electric field may induce a charge, or at least a partialcharge, on a fluidic droplet surrounded by a liquid. In some cases, thefluid and the liquid may be present in a channel, microfluidic channel,or other constricted space that facilitates the electric field to beplaced on the field, for example, by limiting movement of the fluidwithin the liquid. The fluid within the fluidic droplet and the liquidmay be essentially immiscible, i.e., immiscible on a time scale ofinterest (e.g., the time it takes a fluidic droplet to flow through aparticular system or device). In some cases, the fluid may contain otherentities, for example, certain molecular species (e.g., as furtherdiscussed below), cells (e.g., encapsulated by the fluid), particles,etc. In one embodiment, the fluid is present as a series of fluidicdroplets within the liquid.

If the liquid contains a series of fluidic droplets within the liquid,in one set of embodiments, the series of droplets may have asubstantially homogenous distribution of diameters, e.g., the dropletsmay have a distribution of diameters in some cases such that no morethan about 10%, about 5%, about 3%, about 1%, about 0.03%, or about0.01% of the droplets have an average diameter greater than about 10%,about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the averagediameter of the droplets. If more than one series of fluidic droplets isused (e.g., arising from two different sources), each of the series may,in some cases, have a substantially homogenous distribution ofdiameters, although the average diameters of the fluids within eachseries do not necessarily have to be the same.

In another set of embodiments, a charge or partial charge on one or bothdroplets may be induced that causes the two droplets to fuse orcoalesce. Electronic charge may be placed on fluidic droplets within aliquid using any suitable technique, for example, by placing the fluidwithin an electric field, as previously discussed, or by causing areaction to occur that causes the fluid to have an electric charge, forexample, a chemical reaction, an ionic reaction, a photocatalyzedreaction, etc. In one set of embodiments, the fluid within the fluidicdroplet may be an electrical conductor. As used herein, a “conductor” isany material having a conductivity of at least about the conductivity of18 megohm water. The liquid surrounding the fluidic droplet may have anyconductivity less than that of the fluidic droplet, i.e., the liquid maybe an insulator or a “leaky insulator.” In one non-limiting embodiment,the fluidic droplet may be substantially hydrophilic and the liquidsurrounding the fluidic droplet may be substantially hydrophobic.

In one set of embodiments, the charge placed on the fluidic droplet maybe at least about 10⁻²² C/micrometer³. In certain cases, about thecharge may be at least about 10⁻²¹ C/micrometer³, and in other cases,the charge may be at least about 10⁻²⁰ C/micrometer³, at least about10⁻¹⁹ C/micrometer³, at least about 10⁻¹⁸ C/micrometer³, at least about10⁻¹⁷ C/micrometer³, at least about 10⁻¹⁶ C/micrometer³, at least about10⁻¹⁵ C/micrometer³, at least about 10⁻¹⁴ C/micrometer³, at least about10⁻¹³ C/micrometer³, at least about 10⁻¹² C/micrometer³, at least about10⁻¹¹ C/micrometer³, at least about 10⁻¹⁰ C/micrometer³, or at leastabout 10⁻⁹ C/micrometer³ or more. In another set of embodiments, thecharge placed on the fluidic droplet may be at least about 10⁻²¹C/micrometer² (surface area of the fluidic droplet), and in some cases,the charge may be at least about 10⁻²⁰ C/micrometer², at least about10⁻¹⁹ C/micrometer², at least about 10⁻¹⁸ C/micrometer², at least about10⁻¹⁷ C/micrometer², at least about 10⁻¹⁶ C/micrometer², at least about10⁻¹⁵ C/micrometer², at least about 10⁻¹⁴ C/micrometer², or at leastabout 10⁻¹³ C/micrometer² or more. In yet another set of embodiments,the charge may be at least about 10⁻¹⁴ C/droplet, and, in some cases, atleast about 10⁻¹³ C/droplet, in other cases at least about 10⁻¹²C/droplet, in other cases at least about 10⁻¹¹ C/droplet, in other casesat least about 10⁻¹⁰ C/droplet, or in still other cases at least about10⁻⁹ C/droplet.

Additionally, due to the electronic nature of the electric field, veryrapid coalescence and/or reaction speeds may be achieved, according tosome embodiments of the invention. For example, at least about 10droplets per second may be fused or coalesced, and in other cases, atleast about 20 droplets per second, at least about 30 droplets persecond, at least about 100 droplets per second, at least about 200droplets per second, at least about 300 droplets per second, at leastabout 500 droplets per second, at least about 750 droplets per second,at least about 1000 droplets per second, at least about 1500 dropletsper second, at least about 2000 droplets per second, at least about 3000droplets per second, at least about 5000 droplets per second, at leastabout 7500 droplets per second, at least about 10,000 droplets persecond, at least about 15,000 droplets per second, at least about 20,000droplets per second, at least about 30,000 droplets per second, at leastabout 50,000 droplets per second, at least about 75,000 droplets persecond, at least about 100,000 droplets per second, at least about150,000 droplets per second, at least about 200,000 droplets per second,at least about 300,000 droplets per second, at least about 500,000droplets per second, at least about 750,000 droplets per second, atleast about 1,000,000 droplets per second, at least about 1,500,000droplets per second, at least about 2,000,000 or more droplets persecond, or at least about 3,000,000 or more droplets per second may befused or coalesced.

In addition, the electric field can be readily activated or deactivated,applied to a certain number or percentage of the fluidic droplets, orthe like. Furthermore, the coalescence of the fluidic droplets can occurat a specific, predetermined time, and/or location within a channel. Forexample, a chemical reaction may occur (and/or cease to occur) once afirst fluidic droplet and a second fluidic droplet coalesce or fuse.

In one set of embodiments, fluidic droplets to be fused or coalescedneed not be the same size or have the same volume or diameter, accordingto another set of embodiments. For example, a first droplet (e.g., froma first series of droplets) may have a volume greater a second fluidicdroplet (e.g., from a second series of droplets), for instance, suchthat the first droplet has an average diameter that is greater thanabout 125% of the second droplet, and in some cases, greater than about150%, greater than about 200%, greater than about 300%, greater than400%, greater than 500%, etc., relative to the second droplet.

It should be noted, however, that when two droplets “coalesce,” perfectmixing of the two droplets does not instantaneously occur. Instead, acombined droplet may initially be formed of a first region (from a firstdroplet) and a second region (from a second droplet). In some cases, thetwo regions may remain as separate regions, thus resulting in anon-uniform fluid droplet, e.g., if the first fluidic droplet and thesecond fluidic droplet each have a different composition. In some cases,the two regions within the droplet may remain separate (withoutadditional mixing factors) due to the flow of fluid within the droplet.The droplet may also exhibit internal “counter-revolutionary” flow,which may prevent the two fluids from substantially mixing in somecases.

However, in other cases, the two regions within the combined droplet maybe allowed to mix, react, or otherwise interact with each other,resulting in a homogeneously (i.e., completely) mixed, or at leastpartially mixed, fluid droplet. The mixing may occur through naturalprocesses, for example, through diffusion (e.g., through the interfacebetween the two regions), through reaction of the two fluids with eachother, or through fluid flow within the droplet (i.e., convection).However, in some cases, mixing within the fluidic droplet may beenhanced in some fashion. For example, the droplet may be passed throughone or more regions which cause the droplet to change direction in somefashion. The change of direction may alter convection patterns withinthe droplet, allowing the two fluids to be mixed, resulting in an atleast partially mixed droplet.

In one set of embodiments, coalescence of two (or more) fluidic dropletsmay be used to control a reaction involving one or more reactantscontained within one or more of the fluidic droplets. As one example, afirst fluidic droplet may contain a first reactant and a second fluidicdroplet may contain a second reactant, where a reaction occurs when thefirst reactant and the second reactant come into contact. Thus, prior tocoalescence of the first and second fluidic droplets, the first andsecond reactants are not in direct contact and are thus unable to react.After coalesce, e.g., by application of an electric field, the first andsecond reactants come into contact and the reaction may proceed. Thus,the reaction may be controlled, for example, such that the reactionoccurs at a certain time and/or at a certain point within a channel,e.g., as determined by an applied electric field. If the reaction isdeterminable in some fashion (e.g., using a color change), the reactionmay be determined as a function of time, or distance traveled in thechannel.

As another example, one or both droplets may be a cell. For example, ifboth droplets are (or contain) cells, the two cells may be fusedtogether, for example, to create a hybridoma. In another example, onedroplet may be a cell and the other droplet may contain an agent to bedelivered to the cell, for example, a nucleic acid (e.g., DNA, forexample, for gene therapy), a protein, a hormone, a virus, a vitamin, anantioxidant, etc.

As yet another example, one of the two droplets to be fused or coalescedmay contain an ongoing chemical reaction (e.g., of an enzyme and asubstrate), while the other droplet contains an inhibitor to thechemical reaction, which may partially or totally inhibit the reaction,for example, due to competitive or noncompetitive inhibition (i.e., thesecond reactant reacts with the first reactant, inhibiting the firstreactant from participating in other reactions). Thus, coalescence ofthe droplets may inhibit the ongoing chemical reaction, e.g., partiallyor totally. In some embodiments, additional reactions and/or other stepsmay be performed on the coalesced droplet, before or after mixing of thetwo original droplets.

The reaction may be very tightly controlled in some cases. For instance,the fluidic droplets may consist essentially of a substantially uniformnumber of entities of a species therein (i.e., molecules, cells,particles, etc.). For example, 90%, 93%, 95% 97%, 98%, or 99%, or moreof the droplets may each contain the same number of entities of aparticular species. For instance, a substantial number of the dropletsso produced may each contain 1 entity, 2 entities, 3 entities, 4entities, 5 entities, 7 entities, 10 entities, 15 entities, 20 entities,25 entities, 30 entities, 40 entities, 50 entities, 60 entities, 70entities, 80 entities, 90 entities, 100 entities, etc., where theentities are molecules or macromolecules, cells, particles, etc. In somecases, the droplets may contain a range of entities, for example, lessthan 20 entities, less than 15 entities, less than 10 entities, lessthan 7 entities, less than 5 entities, or less than 3 entities. Thus, bycontrolling the number or amount of reactants within each fluidicdroplet, a high degree of control over the reaction may be achieved.

reaction, in one embodiment, may be a precipitation reaction (e.g., thetwo or more reactants may react to produce a particle, for example, aquantum dot). The two reactants may also be, for example, two reactivechemicals, two proteins, an enzyme and a substrate, two nucleic acids, aprotein and a nucleic acid, an acid and a base, an antibody and anantigen, a ligand and a receptor, a chemical and a catalyst, etc.

In one aspect of the present invention, emulsions are formed by flowingtwo, three, or more fluids through a system of conduits. The system maybe a microfluidic system. “Microfluidic,” as used herein, refers to adevice, apparatus or system including at least one fluid channel havinga cross-sectional dimension of less than about 1 millimeter (mm), and insome cases, a ratio of length to largest cross-sectional dimension of atleast 3:1. One or more conduits of the system may be a capillary tube.In some cases, multiple conduits are provided, and in some embodiments,at least some are nested, as described herein. The conduits may be inthe microfluidic size range and may have, for example, average innerdiameters, or portions having an inner diameter, of less than about 1millimeter, less than about 300 micrometers, less than about 100micrometers, less than about 30 micrometers, less than about 10micrometers, less than about 3 micrometers, or less than about 1micrometer, thereby providing droplets having comparable averagediameters. One or more of the conduits may (but not necessarily), incross section, have a height that is substantially the same as a widthat the same point. Conduits may include an orifice that may be smaller,larger, or the same size as the average diameter of the conduit. Forexample, conduit orifices may have diameters of less than about 1 mm,less than about 500 micrometers, less than about 300 micrometers, lessthan about 200 micrometers, less than about 100 micrometers, less thanabout 50 micrometers, less than about 30 micrometers, less than about 20micrometers, less than about 10 micrometers, less than about 3micrometers, etc. In cross-section, the conduits may be rectangular orsubstantially non-rectangular, such as circular or elliptical. Theconduits of the present invention can also be disposed in or nested inanother conduit, and multiple nestings are possible in some cases. Insome embodiments, one conduit can be concentrically retained in anotherconduit and the two conduits are considered to be concentric. In otherembodiments, however, one conduit may be off-center with respect toanother, surrounding conduit. By using a concentric or nesting geometry,the inner and outer fluids, which are typically miscible, may avoidcontact, which can facilitate great flexibility in making multipleemulsions and in devising techniques for encapsulation and polymerosomeformation. For example, this technique allows for fabrication ofcore-shell structure, and these core-shell structures can be convertedinto capsules.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs flow of a fluid. The channelcan have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and/or outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, 10:1,15:1, 20:1, or more. An open channel generally will includecharacteristics that facilitate control over fluid transport, e.g.,structural characteristics (an elongated indentation) and/or physical orchemical characteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

The channel may be of any size, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm or 2 mm, or lessthan about 1 mm, or less than about 500 microns, less than about 200microns, less than about 100 microns, less than about 60 microns, lessthan about 50 microns, less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.In some cases the dimensions of the channel may be chosen such thatfluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art. Insome cases, more than one channel or capillary may be used. For example,two or more channels may be used, where they are positioned inside eachother, positioned adjacent to each other, positioned to intersect witheach other, etc.

As the systems described herein may be three-dimensional microfluidicdevices, e.g., having concentric conduit arrangements, a fluid (of anynesting level of a multiple emulsion) can be completely shielded from asurrounding fluid in certain embodiments. This may reduce or eliminateproblems that can occur in other systems, when the fluids may contacteach other at or near a solid surface, such as in a two-dimensionalsystem.

In some embodiments, a flow pathway can exist in an inner conduit and asecond flow pathway can be formed in a coaxial space between theexternal wall of the interior conduit and the internal wall of theexterior conduit, as discussed in detail below. The two conduits may beof different cross-sectional shapes in some cases. In one embodiment, aportion or portions of an interior conduit may be in contact with aportion or portions of an exterior conduit, while still maintaining aflow pathway in the coaxial space. Different conduits used within thesame device may be made of similar or different materials. For example,all of the conduits within a specific device may be glass capillaries,or all of the conduits within a device may be formed of a polymer, forexample, polydimethylsiloxane, as discussed below.

A geometry that provides coaxial flow can also provide hydrodynamicfocusing of that flow, according to certain embodiments of theinvention. Many parameters of the droplets, including any suitablenesting layer in a multiple emulsion droplet, can be controlled usinghydrodynamic focusing. For instance, droplet diameter, outer dropletthickness and the total number of inner droplets per droplet can becontrolled. Parameters for controlling emulsion or droplet formation canbe controlled by adjusting, for example, the system geometry, and/or theflowrate of any of the fluids used to form the emulsion or droplet.

A variety of materials and methods, according to certain aspects of theinvention, can be used to form systems, such as microfluidic systems,(such as those described above) able to produce the droplets describedherein. In some cases, the various materials selected lend themselves tovarious methods. For example, various components of the invention can beformed from solid materials, in which the channels can be formed viamicromachining, film deposition processes such as spin coating andchemical vapor deposition, laser fabrication, photolithographictechniques, etching methods including wet chemical or plasma processes,and the like. See, for example, Scientific American, 248:44-55, 1983(Angell, et al). In one embodiment, at least a portion of the fluidicsystem is formed of silicon by etching features in a silicon chip.Technologies for precise and efficient fabrication of various fluidicsystems and devices of the invention from silicon are known. In anotherembodiment, various components of the systems and devices of theinvention can be formed of a polymer, for example, an elastomericpolymer such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene(“PTFE” or Teflon®), or the like.

Different components can be fabricated of different materials. Forexample, a base portion including a bottom wall and side walls can befabricated from an opaque material such as silicon or PDMS, and a topportion can be fabricated from a transparent or at least partiallytransparent material, such as glass or a transparent polymer, forobservation and/or control of the fluidic process. Components can becoated so as to expose a desired chemical functionality to fluids thatcontact interior channel walls, where the base supporting material doesnot have a precise, desired functionality. For example, components canbe fabricated as illustrated, with interior channel walls coated withanother material. Material used to fabricate various components of thesystems and devices of the invention, e.g., materials used to coatinterior walls of fluid channels, may desirably be selected from amongthose materials that will not adversely affect or be affected by fluidflowing through the fluidic system, e.g., material(s) that is chemicallyinert in the presence of fluids to be used within the device.

In one embodiment, various components of the invention are fabricatedfrom polymeric and/or flexible and/or elastomeric materials, and can beconveniently formed of a hardenable fluid, facilitating fabrication viamolding (e.g. replica molding, injection molding, cast molding, etc.).The hardenable fluid can be essentially any fluid that can be induced tosolidify, or that spontaneously solidifies, into a solid capable ofcontaining and/or transporting fluids contemplated for use in and withthe fluidic network. In one embodiment, the hardenable fluid comprises apolymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).Suitable polymeric liquids can include, for example, thermoplasticpolymers, thermoset polymers, or mixture of such polymers heated abovetheir melting point. As another example, a suitable polymeric liquid mayinclude a solution of one or more polymers in a suitable solvent, whichsolution forms a solid polymeric material upon removal of the solvent,for example, by evaporation. Such polymeric materials, which can besolidified from, for example, a melt state or by solvent evaporation,are well known to those of ordinary skill in the art. A variety ofpolymeric materials, many of which are elastomeric, are suitable, andare also suitable for forming molds or mold masters, for embodimentswhere one or both of the mold masters is composed of an elastomericmaterial. A non-limiting list of examples of such polymers includespolymers of the general classes of silicone polymers, epoxy polymers,and acrylate polymers. Epoxy polymers are characterized by the presenceof a three-membered cyclic ether group commonly referred to as an epoxygroup, 1,2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Non-limiting examples of siliconeelastomers suitable for use according to the invention include thoseformed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred in one set of embodiments, for example,the silicone elastomer polydimethylsiloxane. Non-limiting examples ofPDMS polymers include those sold under the trademark Sylgard by DowChemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184,and Sylgard 186. Silicone polymers including PDMS have severalbeneficial properties simplifying fabrication of the microfluidicstructures of the invention. For instance, such materials areinexpensive, readily available, and can be solidified from aprepolymeric liquid via curing with heat. For example, PDMSs aretypically curable by exposure of the prepolymeric liquid to temperaturesof about, for example, about 65° C. to about 75° C. for exposure timesof, for example, about an hour. Also, silicone polymers, such as PDMS,can be elastomeric, and thus may be useful for forming very smallfeatures with relatively high aspect ratios, necessary in certainembodiments of the invention. Flexible (e.g., elastomeric) molds ormasters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus, components can be fabricated and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention, as well as overall molding techniques, are described in theart, for example, in an article entitled “Rapid Prototyping ofMicrofluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480,1998 (Duffy, et al.), incorporated herein by reference.

In some embodiments, certain microfluidic structures of the invention(or interior, fluid-contacting surfaces) may be formed from certainoxidized silicone polymers. Such surfaces may be more hydrophilic thanthe surface of an elastomeric polymer. Such hydrophilic channel surfacescan thus be more easily filled and wetted with aqueous solutions.

In one embodiment, a bottom wall of a microfluidic device of theinvention is formed of a material different from one or more side wallsor a top wall, or other components. For example, the interior surface ofa bottom wall can comprise the surface of a silicon wafer or microchip,or other substrate. Other components can, as described above, be sealedto such alternative substrates. Where it is desired to seal a componentcomprising a silicone polymer (e.g. PDMS) to a substrate (bottom wall)of different material, the substrate may be selected from the group ofmaterials to which oxidized silicone polymer is able to irreversiblyseal (e.g., glass, silicon, silicon oxide, quartz, silicon nitride,polyethylene, polystyrene, epoxy polymers, and glassy carbon surfaceswhich have been oxidized). Alternatively, other sealing techniques canbe used, as would be apparent to those of ordinary skill in the art,including, but not limited to, the use of separate adhesives, thermalbonding, solvent bonding, ultrasonic welding, etc.

The following applications are each incorporated herein by reference:U.S. patent application Ser. No. 08/131,841, filed Oct. 4, 1993,entitled “Formation of Microstamped Patterns on Surfaces and DerivativeArticles,” by Kumar, et al., now U.S. Pat. No. 5,512,131, issued Apr.30, 1996; U.S. patent application Ser. No. 09/004,583, filed Jan. 8,1998, entitled “Method of Forming Articles including Waveguides viaCapillary Micromolding and Microtransfer Molding,” by Kim, et al., nowU.S. Pat. No. 6,355,198, issued Mar. 12, 2002; International PatentApplication No. PCT/US96/03073, filed Mar. 1, 1996, entitled“Microcontact Printing on Surfaces and Derivative Articles,” byWhitesides, et al., published as WO 96/29629 on Jun. 26, 1996;International Patent Application No.: PCT/US01/16973, filed May 25,2001, entitled “Microfluidic Systems including Three-DimensionallyArrayed Channel Networks,” by Anderson, et al., published as WO 01/89787on Nov. 29, 2001; U.S. patent application Ser. No. 11/246,911, filedOct. 7, 2005, entitled “Formation and Control of Fluidic Species,” byLink, et al., published as U.S. Patent Application Publication No.2006/0163385 on Jul. 27, 2006; U.S. patent application Ser. No.11/024,228, filed Dec. 28, 2004, entitled “Method and Apparatus forFluid Dispersion,” by Stone, et al., published as U.S. PatentApplication Publication No. 2005/0172476 on Aug. 11, 2005; InternationalPatent Application No. PCT/US2006/007772, filed Mar. 3, 2006, entitled“Method and Apparatus for Forming Multiple Emulsions,” by Weitz, et al.,published as WO 2006/096571 on Sep. 14, 2006; U.S. patent applicationSer. No. 11/360,845, filed Feb. 23, 2006, entitled “Electronic Controlof Fluidic Species,” by Link, et al., published as U.S. PatentApplication Publication No. 2007/000342 on Jan. 4, 2007; U.S. patentapplication Ser. No. 11/368,263, filed Mar. 3, 2006, entitled “Systemsand Methods of Forming Particles,” by Garstecki, et al.; andInternational Patent Application No. PCT/US2007/017617, filed Aug. 7,2007, entitled “Fluorocarbon Emulsion Stabilizing Surfactants,” byWeitz, et al., published as WO 2008/021123 on Feb. 21, 2008. Alsoincorporated herein by reference is U.S. Provisional Patent ApplicationSer. No. 60/905,567, filed Mar. 7, 2007, entitled “Assay and OtherReactions Involving Droplets,” by J. J. Agresti, et al.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

Recently, several microfluidic methods have provided a very promisingapproach to prepare monodisperse polymeric microspheres, because suchmicrofluidic methods can control the fluid flow precisely and thusensure high monodispersity of the prepared particles, furthermore, boththe emulsification and the polymerization can be carried out in the samedevice in some cases.

In this example, an on-chip fabricating technique is presented for thepreparation of highly monodisperse and homogenous thermo-sensitivePNIPAM microgels. Instead of using UV irradiation, a redox reactionapproach was used in a microfluidic chip to initiate the polymerizationof N-isopropylacrylamide (NIPAM) monomer. Because the polymerization wascarried out in the chip below the LCST (Lower Critical SolutionTemperature), the resultant PNIPAM microgels were highly homogeneous.Another advantage of this approach is that all fabrication processeswere completed in one chip and without any other supplementaryinstruments, e.g., a UV lamp; thus, this microfluidic reactor is morecompact, which makes it more easily scalable.

The microfluidic chip in this example was made of poly(dimethylsiloxane)(PDMS) by using a standard soft-lithography method known to those ofordinary skill in the art, which allows rapid replication of anintegrated microchannel prototype. Flow-focusing geometry was used inthis device to generate monodisperse emulsion droplets, as shown inFIG. 1. In particular, FIG. 1A shows a schematic illustration of thechannel design in the microfluidic chip. FIG. 1B is a photograph of thePDMS microfluidic system compared with a one-dime coin of the USA. Thepolyethylene tubing and microchannels were filled with a dye-labeledaqueous solution to increase the contrast of this image. The channels inthe PDMS device have a height of 15 micrometer. The throat channel has awidth of 10 micrometer and a length of 270 micrometer. Fluid 1 is anaqueous solution containing monomer, initiator and crosslinker, and thisfluid was pumped through an inlet channel with width of 20 micrometer.The viscosity of the fluid 1 was about 1 mPa s. Fluid 2 was a kerosenesolution containing surfactant, and this fluid was pumped through twoflanking channels with a width of 30 micrometer as the continuous phase.The viscosity of the fluid 2 was about 4 mPa·s. Fluid 3 was a kerosenesolution containing surfactant and accelerator.

In the emulsification-polymerization approach to prepare PNIPAMhydrogels, the polymerization of NIPAM monomer was usually initiated byadding accelerator into the monomer solution to start the redoxreaction, or by heating the monomer solution to a temperature above theLCST or by irradiating with UV light. Generally, the redox reactiongenerated homogeneous internal microstructure inside the hydrogel, whilethe latter two methods resulted in heterogeneous internalmicrostructure. The PNIPAM microgel with homogeneous microstructure mayhave a much larger thermo-responsive volume-change ratio than that withheterogeneous microstructure. To prepare homogeneous microgels in themicrofluidic chip, if the accelerator is added into the monomersolution, the polymerization may occur in a very short time; thus, themicrochannel can be clogged by the polymerized PNIPAM hydrogels and nomicrogels may form. To prevent this, here a novel approach was used toadd the accelerator. The accelerator was put in the oil phase and addedinto the downstream emulsion solution (FIG. 1). Because the acceleratorN,N,N′,N′-tetramethylethylenediamine (TEMED) is more soluble in water;when it is added into the oil phase (FIG. 1D), it will diffuse into thewater phase. FIG. 1D is an optical microscope image of addingaccelerator solution in the downstream of emulsification. When theaccelerator meets the initiator ammonium persulfate (APS) inside themonomer emulsion droplets, a redox reaction is initiated to polymerizethe NIPAM monomer. Thus, not only the channel clogging problem wasavoided or at least reduced, but also, homogeneous internalmicrostructure was generated inside the microgels.

In the system shown in FIG. 1, fluid 1 was an aqueous solutioncontaining monomer NIPAM (11.3% w/v), an initiator APS (1.13% w/v) and acrosslinker N,N′-methylenebisacryamide (BIS, 0.77% w/v); fluid 2 was akerosene solution containing surfactant polyglycerol polyricinoleate(PGPR 90, Danisco, 8% w/v); and fluid 3 was a kerosene solutioncontaining both PGPR 90 (8% w/v) and an accelerator TEMED (10% v/v). Thesolutions were supplied to the microfluidic device through polyethylenetubing (Scientific Commodities) attached to syringes (Hamilton Gastight)operated by syringe pumps (Harvard Apparatus, PHD 2000 series). APhantom high-speed camera (Vision Research) was used to record the dropformation processes.

One feature of this microfluidic approach is that precise control of thedrop sizes inside the channel could be achieved while maintaining sizemonodispersity. A thin and long throat channel was used for the dropformation (FIG. 1C). FIG. 1C is an optical microscope image of the dropformation in the emulsification step. In these experiments, monodispersemonomer droplets were generated over the size range from about 10 toabout 3 micrometers by radius, as shown in FIG. 2. For given viscositiesof fluids and geometry of the device, controlling the flow rate ratio,Q_(CF)/Q_(DF), which is the relative flow rate of the continuous fluid,Q_(CF), to the dispersed fluid, Q_(DF), gave rise to different dropsizes. When Q_(CF)/Q_(DF) was less than about 4, drop break-up occurredat the end of the throat channel (FIG. 2A) and the resulting drop sizeswere bigger than that of the cross-section dimension of throat channelby factor of about two. FIG. 2A is an optical microscope image of thedrop formation at a low flow rate ratio, Q_(CF)/Q_(DF). In this case,the droplet size was still monodisperse because the solid wall of thethroat channel made the flow inside the channel relatively stable.

As Q_(CF)/Q_(DF) was increased, smaller drop sizes were created.Moreover, when Q_(CF)/Q_(DF) was higher than about 6, the external flowcreated larger drag force, thus forming drops which were smaller thanthe dimension of throat channel (FIG. 2B). FIG. 2B is an opticalmicroscope image of the drop formation at a high Q_(CF)/Q_(DF). Thescale bar is 25 micrometer. Owing to the presence of the surfactant, inthis study, the coalescence of the drops was prevented as they floweddown the streams. However, no matter where drop formation occurred,e.g., at the entrance or at the exit of the throat channel, the monomerdroplets were highly monodisperse with standard deviations less thanabout 2%.

FIG. 2C shows dependence of drop radii on Q_(CF)/Q_(DF). Q_(CF)/Q_(DF)was controlled by fixing the Q_(DF) with 30 microliter h⁻¹ and changingthe Q_(CF) from 90 microliter h⁻¹ to 320 microliter h⁻¹. Open circlescorrespond to the case of FIG. 2A; and closed circles correspond to thecase of FIG. 2B.

After polymerization, the microgels were washed 5 times with isopropanolby centrifuge (IEC Centra, CL2; 3000 rpm, 2 min for each wash) to removethe oil on the microgel surface, and then immersed into pure water. Totest the thermo-sensitive volume-phase transition behavior, the PNIPAMmicrogels together with pure water were put into a transparent sealedholder on the slide glass, which was put on a heating and cooling stagefor microscope (Physitemp Instruments, TS-4ER). The actual temperatureinside the sample holder was measured by an infrared thermometer. Adigital camera (Hamamatsu, C4742-95) was used to record thethermo-responsive behavior of microgels. The prepared PNIPAM microgelsillustrated homogeneous structures (FIG. 3A) and excellentthermo-sensitivity. At 22° C., the diameter of the PNIPAM microgels wasabout 2.7 times as that at 40° C. (FIG. 3). FIG. 3A is an opticalmicroscope image of the microgels in pure water at 22° C. FIG. 3B is anoptical microscope image of the same microgels in pure water at 40° C.The scale bar is 25 micrometers. The temperature-dependent diameterchange of the microgel was found to be generally reversible and showed asharp transition near the LCST, as shown in FIG. 3C.

In summary, this example shows a facile approach to prepare monodispersethermo-sensitive microgels in the microfluidic chip. The preparationprocesses are all done in one chip. This method was compact andscalable. By using this technique, PNIPAM microgels were obtained withhighly monodispersity with less than about 2% standard deviations, andhaving homogenous internal microstructures as well as excellentthermo-sensitivities, which may be important properties for themicrogels to be efficiently used in some cases. This approach can alsobe used to prepare other polymeric microspheres, e.g., by initiating aredox reaction to cause polymerization. Furthermore, this method canalso be easily used to prepare multi-functional microgels with smallsize ranges by incorporating functional substances into the monomersolution.

Example 2

This example illustrates the probing of DNA sequences in hydrogelparticles. A PDMS microfluidic device similar to that described inExample 2 was made from an SU8 on Si mold using standard softlithography methods. In these experiments, two populations of gelparticles containing acrydite-coupled template DNA, differing onlyslightly, were probed with fluorescent DNA oligonucleotides. The twoprobes were prepared with different fluorescent species, Alexa-488(green) and Cy-5 (red).

Initially, a mixture of two different populations of gel particles(labeled B and E), each incorporating a different fragment of theacrydite-labeled DNA, were washed three times in an annealing buffer (50mM NaCl, 1.5 mM MgCl₂, Tris.HCl, pH 8.0). The B-population fragment usedin the gel particles had the sequenceacrydite-TCGCGGTTTCGCTGCCCTTTGTTCTCTCCATTGTAGCACGTGTGTAGC CCA (SEQ IDNO: 1), and the E-population fragment sequence wasacrydite-TCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGC CCT (SEQ IDNO: 2).

Next, hybridization probes that were complementary to both B (green,Alexa-488-AGAACAAAGGGCAGCGAAAC, SEQ ID NO: 3) and to E (red,Cy-5-CATACAAAGAGAAGCGACCTCG, SEQ ID NO: 4) were added to the particlesuspensions at a concentration of 500 nM. The samples were heated to 95°C. and cooled slowly to 57° C., then held at 57° C. for 10 min. Thetemperature was then decreased to 53° C., and 50 ml of 53° C. washbuffer was added (0.1 M Tris-C1, pH 7.5, 20 mM EDTA, 0.5 M KCl). Thereaction was then washed twice further with 1 ml of wash buffer. Thelabeled gels were imaged with a fluorescence microscope.

FIGS. 4A and 4B illustrate brightfield and fluorescence images,respectively, of gel droplets prepared that contained one of the two DNAsequences. As mentioned above, the two populations, containing thedifferent polymorphisms, were mixed together. Fluorescence imaging ofthe red and green dyes were used to identify the polymorphisms as shownin FIG. 4B (lighter droplets are “green,” while darker droplets are“red,” in this figure).

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method of amplification comprising: a.generating a first plurality of droplets comprising polynucleotideprimers, wherein the first plurality of droplets excludespolynucleotides to be amplified; b. generating a second plurality ofdroplets comprising polynucleotides to be amplified, wherein thepolynucleotides are obtained from a whole genome; c. merging firstdroplets of the first plurality of droplets with second droplets of thesecond plurality of droplets to form coalesced droplets; and d.performing amplification reactions on the polynucleotides in thecoalesced droplets to form amplicons.
 2. The method of claim 1, whereinthe amplification reactions comprise amplifying a plurality of differentregions of the polynucleotides obtained from the whole genome.
 3. Themethod of claim 1, wherein the amplification reaction is performed usingPhi29 polymerase.
 4. The method of claim 1, wherein the whole genomecomprises a polymorphism.
 5. The method of claim 1, wherein the wholegenome is a mammalian genome.
 6. The method of claim 1, wherein thewhole genome is a primate genome.
 7. The method of claim 1, wherein thepolynucleotides obtained from the whole genome are DNA.
 8. The method ofclaim 1, wherein the polynucleotides obtained from the whole genome areRNA.
 9. The method of claim 1, wherein the first plurality of dropletsfurther comprise amplification reagents.
 10. The method of claim 1,wherein the second plurality of droplets further comprise amplificationreagents.
 11. The method of claim 1, further comprising sequencing theamplicons.
 12. The method of claim 1, wherein the amplificationreactions are selected from the group consisting of: Polymerase ChainReaction (PCR), reverse-transcription PCR and real-time PCR.
 13. Themethod of claim 12, wherein the amplification reactions are reversetranscription PCR.
 14. The method of claim 1, wherein the polynucleotideprimers comprise an acrydite moiety.
 15. The method of claim 1, furthercomprising hardening the coalesced droplets to form gels.
 16. The methodof claim 15, wherein the polynucleotide primers are bound to the gels.17. The method of claim 15, wherein the hardening comprises introducinga gelation initiator.
 18. The method of claim 17, wherein the gelationinitiator co-flows with an aqueous phase.
 19. The method of claim 17,wherein the gelation initiator co-flows with an oil phase.
 20. Themethod of claim 17, wherein the gelation initiator is selected from thegroup consisting of ammonium persulfate, TEMED and Ca²⁺.
 21. The methodof claim 15, wherein the gels comprise poly(acrylamide).
 22. The methodof claim 1, wherein the method has a rate of coalescence of at least 10droplets per second.
 23. The method of claim 1, wherein the averagediameter of the first, second or coalesced droplets is at least 1micrometer.
 24. The method of claim 1, wherein the first plurality ofdroplets comprises a plurality of identical polynucleotide primers. 25.The method of claim 1, wherein some of the droplets in the firstplurality of droplets are distinct.
 26. The method of claim 1, whereinthe amplification reactions are performed using primers from the firstdroplets.
 27. The method of claim 1, wherein the merging of the firstdroplets with the second droplets is controlled.
 28. The method of claim27, wherein the merging of the first droplets with the second dropletsis electrically controlled.
 29. The method of claim 17, wherein thegelation initiator comprises a reducing agent.
 30. The method of claim1, wherein the first, second or coalesced droplets comprise anidentifier.
 31. The method of claim 1, wherein droplets within the firstplurality of droplets comprise different identifiers.
 32. The method ofclaim 31, wherein the identifier is an optical identifier.
 33. Themethod of claim 31, wherein the identifier is a fluorophore.
 34. Themethod of claim 1, wherein the first plurality of droplets or the secondplurality of droplets comprise aqueous droplets within an oil phase. 35.The method of claim 34, wherein the oil phase comprises fluorocarbonoil.
 36. The method of claim 35, wherein the fluorocarbon oil comprisesKrytox.
 37. The method of claim 1, wherein the first, second orcoalesced droplets comprise a fluoro surfactant.
 38. The method of claim1, wherein the first, second or coalesced droplets comprise an ammoniumcarboxylate salt surfactant.
 39. The method of claim 1, wherein thefirst, second or coalesced droplets comprise a surfactant selected fromthe group consisting of Span80, Span80/Tween-20, Span80/Triton X-100,Abil EM90, Abil we09, polyglycerol polyricinoleate “PGPR90”, Tween-85,749 Fluid, and ammonium carboxylate salt of Krytox.